As such, a candidate SARS vaccine should elicit broad CD8+ T-cell immune responses.. A high level of specific SARS-CD8+ T-cell response was demonstrated in mice that received DNA encodin
Trang 1Open Access
Research
A combined nucleocapsid vaccine induces vigorous SARS-CD8+
T-cell immune responses
Ali Azizi1,2, Susan Aucoin1, Helina Tadesse2, Rita Frost1, Masoud Ghorbani1, Catalina Soare1,2, Turaya Naas1,2 and Francisco Diaz-Mitoma*1,2
Address: 1 Infectious Disease and Vaccine Research Centre, Children's Hospital of Eastern Ontario Research Institute, 401 Smyth Road, Ottawa,
ON, K1H 8L1, Canada and 2 Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, K1H 8M2, Canada
Email: Ali Azizi - aliazizi555@yahoo.ca; Susan Aucoin - saucoin@cheo.on.ca; Helina Tadesse - htade096@uottawa.ca;
Rita Frost - frost@cheo.on.ca; Masoud Ghorbani - mghorbani@yahoo.com; Catalina Soare - catalinasoare@yahoo.com;
Turaya Naas - turayanaas@hotmail.com; Francisco Diaz-Mitoma* - diaz@cheo.on.ca
* Corresponding author
VaccineSARSNucleocapsidXIAP
Abstract
Several studies have shown that cell-mediated immune responses play a crucial role in controlling
viral replication As such, a candidate SARS vaccine should elicit broad CD8+ T-cell immune
responses Several groups of mice were immunized alone or in combination with
SARS-nucleocapsid immunogen A high level of specific SARS-CD8+ T-cell response was demonstrated
in mice that received DNA encoding the SARS-nucleocapsid, protein and XIAP as an adjuvant We
also observed that co-administration of a plasmid expressing nucleocapsid, recombinant protein
and montanide/CpG induces high antibody titers in immunized mice Moreover, this vaccine
approach merits further investigation as a potential candidate vaccine against SARS
Introduction
The SARS epidemic had a high mortality rate as well as a
huge economic impact worldwide Treatment with
antivi-ral drugs or an effective vaccine is not available for
protec-tion against this disease [1,2] The SARS-CoV is a
single-stranded RNA virus that has been identified as a new type
of coronavirus The genome is approximately 30 kb long
and contains four structural proteins: spike, envelope,
matrix and nucleocapsid in the same order as other
coro-naviruses [3,4] However, the sequence analysis of
SARS-CoV with other members of the coronavirus family did
not show more than 20% nucleotide homology [5]
The SARS-NC gene encodes a 46 kDa protein that partici-pates in the replication and transcription of the virus and interferes with the cell cycle of host cells [6] Previous studies in other coronavirus members suggest that this protein is highly immunogenic and could be a good target for the design of an effective vaccine [7-10] The expres-sion of NC in CHO cells led to the observation that this protein folds spontaneously into viral-like particles (VLPs) These particles are effectively incorporated at sev-eral stages of the virus life cycle, including assembly, bud-ding from cells, and receptor-binbud-ding leabud-ding to membrane fusion The viral particles also present antigens
Published: 22 August 2005
Genetic Vaccines and Therapy 2005, 3:7 doi:10.1186/1479-0556-3-7
Received: 09 May 2005 Accepted: 22 August 2005 This article is available from: http://www.gvt-journal.com/content/3/1/7
© 2005 Azizi et al; licensee BioMed Central Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Trang 2to the immune system in a structure that mimics the
infec-tious virion [11-13]
DNA vaccines are able to induce both humoral and
cellu-lar immune responses and have demonstrated their
effi-cacy in several experimental models [14,15] There are
several eukaryotic vectors that express recombinant
pro-teins efficiently However, the uptake of antigens and its
presentation are critical elements in DNA vaccination
strategies One strategy to increase the potency of DNA
vaccines is to prolong the survival of antigen presenting
cells (APCs), especially dendritic cells Previous studies
show that survival of dendritic cells is increased in the
presence of anti-apoptotic factors such as XIAP This
approach has results in increased amounts of
antigen-spe-cific CD8+ T cells [16,17]
Specific CD8+T-cells play an important role in the control
of viral infection [18-20] Activation of specific CD8+ cells
results in the secretion of inflammatory cytokines (IFN-γ
and TNF-α) [21] and the synthesis of effector molecules,
such as perforin and granzymes which kills infected cells,
decreasing virus replication and virus load [22,23]
The present study characterizes cellular and humoral
immune responses to SARS-CoV in mice receiving a
DNA-NC construct alone or in combination with protein and
different adjuvants The combination of DNA-NC,
pro-tein and XIAP elicited a significant anti-SARS CD8+ T-cell
response independent of CD4+ T-cell immune responses
Materials and methods
Cell culture
Chinese Hamster Ovary (CHO) cells were grown at 37°C,
5% CO2 in Iscove's Modified Dulbecco's Medium
(IMDM: Sigma, St Louis, MO) and supplemented with
10% Fetal Calf Serum (FCS: Life Technologies, Grand
Island, NY), 100 U/ml penicillin and 100 µg/ml
gentamyin
Construction of DNA plasmids
Total RNA was purified using an RNeasy extraction kit
(Qiagen, Mississauga, Ont) from the lung tissue of an
autopsied patient who died from SARS The full-length
NC (1.2 kb) gene was amplified using specific primers
(forward primer: 5'-ggatccatgtctgataatggaccc-3'; reverse
primer: 5'-gaattcttatgcctgagttgaatc-3') The amplicon was
purified using the QIAquick gel extraction kit (Qiagen)
and cloned into the PCR 2.1 TOPO-TA vector (Invitrogen,
Burlington, Ont) according to the manufacturer's
instruc-tions After plasmid digestion, the 1.2 kb band
corre-sponding to the NC gene was sub-cloned into BamHI-and
EcoRI sites of pVAX-1 which contains a CMV promoter for
high level expression in vivo The fragment was also
sub-cloned into the pEF6-Myc/His (Invitrogen) and pQE
(Qiagen) vectors The pEF6 vector was designed to over produce recombinant proteins in mammalian cell lines and was used to establish a stable cell line by using a resistant blasticidine gene The pQE Tri system vector was used for production of proteins in bacteria These vectors ultimately allow for the purification of the protein by immobilized metal affinity chromatography All expres-sion constructs were confirmed and characterized by restriction enzymes and nucleotide sequence analysis
Expression of recombinant nucleocapsid protein
CHO cells and JM109 bacteria were transfected with pEF6 and pQE vectors containing NC or vector alone In order
to increase and sustain expression of the NC protein, a sta-ble NC expressing CHO cell line was established using blasticidine-supplemented medium Cells were harvested, sonicated and lysed in lysis buffer (25 mM Tris base, 2.5
mM Mercaptoethanol, 1% Triton-X100 and a cocktail of protease inhibitors) Cell pellets were centrifuged and the supernatant was incubated with the TALON metal resin (Clontech, Palo Alto, CA) for one hour After incubation, the mixture of protein-resin was added to the columns and washed three times with 20 bed volumes of Tris-Cl, NaCl (PH 8) The recombinant protein was eluted with
150 mM imidazole
To confirm the proteins, samples were mixed with Lae-mmli loading buffer, boiled for 5 minutes and loaded on
a 10% polyacrylamide gel The proteins were then trans-ferred to a nitrocellulose membrane by electrophoretic transfer The membranes were blocked with 5% dried milk in PBS-Tween 20 (PBS-T) and incubated with 1/3000 dilution of a sera from a SARS patient for three hours at room temperature After washing with PBS-Tween, the blots were incubated with anti-human IgG-HRP conjugate (BioRad, Hercules, CA) for one hour at room temperature After incubation, the blots were washed and incubated with ECL reagent (Santa Cruz Biotechnology, Santa Cruz, CA) for one minute and exposed to X-ray film (Kodak)
Electron Microscopy
CHO cells transfected with either the DNA vector express-ing NC or DNA vector alone were harvested and washed with PBS Pellets were then fixed with 2% glutaraldehyde Cells were rinsed twice in 0.1 M sodium cacodylate buffer
at 4°C The cells were then fixed with 2% Osmium Tetrox-ide for 2 hr at 4°C After washing with distilled water, the cells were dehydrated with increasing concentrations of ethanol and embedded in spur resin Thin sections were stained with uranyl acetate and lead citrate The sections were screened by using a JEOL 1010 Transmission Elec-tron Microscope (TEM)
Trang 3CpG oligodeoxynucleotide
(5'-TCCATGACGTTCCT-GACGTT-3') was provided by Coley (Ottawa, ON)
Mon-tanide ISA-51 mineral oil adjuvant was purchased from
Seppic Inc (Paris, France) The pcDNA3 construct
express-ing 1.5 kb XIAP gene encodexpress-ing an anti-apoptotic gene
product was a kind gift from Dr R.G Korneluk [24]
Animal Immunization
Six to eight week-old female B6/C3/F1 mice (Charles
River, St Constant, PQ) were immunized subcutaneously
at the base of the tail with 50 µg of DNA construct
express-ing the nucleocapsid gene, 5µg of nucleocapsid protein
and 50 µl of montanide ISA-51 (Seppic)/30 µg CpG
(Coley), or 50 µg pcDNA3-XIAP at each vaccination Each
mouse was boosted three times, at one month intervals
Fourteen days after the last boost, the mice were sacrificed
and their spleens and blood was collected for further
test-ing or for long-term storage in cryopreservation medium
Antibody measurement by ELISA
96 well ELISA plates were coated overnight at 4°C with
NC protein, and the wells were washed with PBS
contain-ing 0.05% Tween 20 and then blocked with 1% BSA in
PBS Serially diluted sera was added and incubated for 2 h
at 37°C The plates were washed and incubated for 2 h
with a 1/2000 dilution of a peroxidase-conjugated
affin-ity-purified rabbit anti-mouse secondary antibody
(Bio-Rad, Richmond, CA) The plates were washed three times
and developed with O-phenylendiamine dihydrochloride
(OPD) substrate (Sigma, St Louis, MO) The color
reac-tion was stopped with 1N HCl and absorbance was read
at 490 nm with an ELISA plate reader (Bio-Rad)
Proliferation assay
Splenocytes from immunized mice were resuspended at 2
× 106 cells/ml in RPMI 1640 containing 10% FCS, 50 µM
β-mercaptoethanol and 100 U/ml
penicillin/streptomy-cin A 100 µl aliquot containing 2 × 105 cells was added to
each well of a 96 well plate The NC protein (100 µl at
20µg/ml) was added to each well in triplicate As a
posi-tive control, cells were also stimulated with phorbol
12-myristate 13-acetate and ionomycin (PMA/ION) After 72
h of culture, 1 µCi [3H] thymidine (Amersham, Arlington
Heights, IL) was added to each well Following 16 h of
incubation, cells were harvested onto glass fibre filtermats
and thymidine incorporation was measured with a
Microbeta beta counter (Wallac, Turku, Finland)
Intracellular cytokine staining
Fresh blood and splenocytes from immunized mice were
cultured in IMDM media in the presence of 10 µg/ml
brefeldin A (Sigma) and stimulated in vitro with the NC
protein (10 µg/ml) expressed in bacteria In every
experi-ment, a negative control (without stimulation), positive
control (PMA/ION) and an irrelevant protein (HIV-1 gp120 protein) was included to control for spontaneous production of IFN-γ Sixteen hours after incubation, the cells were washed once (1600 rpm for 5 min) with 3 ml PBS / 2% FCS / 0.01% Azide and surface-stained for 15 min with PE-labeled Ab to mouse CD3, TC-labeled Ab to mouse CD8α or CD4 (Caltag Laboratories, Hornby, ON) The cells were washed as above, fixed and permeabilized using 100 µl each of A and B fixation-permeabilization solution (Caltag Laboratories) The cells were stained intracellularly with anti-mouse IFN-γ FITC-labeled Ab and incubated for 30 min (in the dark) at 4°C Following washing, cells were analyzed by FACScan (Becton Dickin-son, Mississauga, ON) An increase of 0.1% of IFN-gamma producing cells over the unstimulated control was considered as positive response to vaccination
ELISPOT assay
Multiscreen-HTS plates (Millipore, Bedford, MA) were coated with 10 µg/ml of anti-mouse IFN-γ antibody (mAb AN18, Mabtech, Mariemont, OH) in PBS over night at 4°C The plates were then washed with PBS and blocked with IMDM containing 10% FCS and 100 U/ml penicil-lin/streptomycin for 1 h at room temperature The medium were removed and 4 × 105 cell suspension (100 µl/well) including NC SARS protein expressed in bacteria (10 µg/ml) or irrelevant antigens at the same concentra-tion were added and incubated for 30 h at 37°C After
Western blot analysis of recombinant SARS-CoV-NC protein
Figure 1
Western blot analysis of recombinant SARS-CoV-NC pro-tein Lane 1: purified protein from JM 109 cells transfected with pQE vector encoding nucleocapsid gene Lane 2: repre-sents cells transfected with the vector alone The blot was probed with sera from a SARS patient
47.5 kDa
32.5 kDa
Trang 4incubation, cells were removed; plates were washed with
PBS+0.05% Tween 20 and incubated with 1 µg/ml of
biotinylated anti-mouse IFN-γ antibody (mAb
R4-6A2-Biotin, Mabtech) for 2 hr at room temperature After
fur-ther washings, 100 µl/well of 1/2000
Streptavidin-ALP-PQ (Mabtech) in PBS+ 0.5% FCS was added and
incu-bated for 1 hr at room temperature The plates were
washed as above and developed with 100 µl per well
BCIP/NBT alkaline phosphatase (Moss Inc) for 20
min-utes at room temperature The reaction was stopped with
rinsing the plates with tap water The numbers of spots
were analyzed with an ELISPOT reader
Statistical analysis
Results were expressed as mean ± S.D In each experiment
four animals were used per group The t-test was applied
for the statistical analysis of the data The p value equal to
or less than 0.05 was considered significant
Results
Construction of the DNA vectors and expression of SARS-nucleocapsid protein in mammalian and bacteria cells
To increase the potency of the specific immune response, the full-length NC was amplified by RT-PCR and ligated into plasmid pVAX-1 under the control of the human cytomegalovirus promoter For the expression and purifi-cation of the recombinant NC protein in CHO and bacteria cells, the amplified NC gene was also sub-cloned into pEF6-Myc/His and pQE-Tri system vectors To express NC protein, CHO cells and E.coli (JM109) were transfected with pEF6 and pQE vectors encoding the NC gene, respectively To increase the yield of the recom-binant protein, a stable CHO cell line was created using a selective resistant blasticidine gene, allowing for efficient purification of the recombinant protein Cells were har-vested, lysed and the recombinant proteins were purified according to standard methods The expression of the NC protein in transfected cells was verified by western blot-ting (Fig 1) and immunofluorescence staining of CHO
Production of viral-like particles shown by electron microscopy
Figure 2
Production of viral-like particles shown by electron microscopy The CHO cells were transfected with DNA-NC or vector alone Arrows indicate VLPs in the transfected cell lines
Trang 5cells infected with the vector-NC or the vector alone
Anti-body raised in rabbits to the NC protein expressed in
bac-teria reacted strongly in the perinuclear region of the
SARS-NC-CHO cell line (data not shown)
The assembly of NC protein into virus like particles (VLPs)
The CHO cells transfected with pEF6-NC or vector alone
were examined by transmission electron microscopy We
observed bundles of VLP of the same morphology as wild
type particles both inside and outside cells infected with
pEF6-NC However, neither the mock-transfected cells
nor the cells transfected with the vector alone showed
viral-like particles (Fig 2) These observations
demon-strate that our construct expressing the NC protein
synthe-sised sufficient protein within infected cells to facilitate
the formation of VLPs
Detection of antibody titer in mice immunized with the
candidate vaccine combinations
In order to analyze the antibody titer against NC, five
groups of mice were primed and boosted with
SARS-nucleocapsid immunogen alone or in combination Two
weeks after the last boost, sera were collected and anti-body titer was measured by ELISA The group received protein and montanide/CpG showed a higher mean IgG antibody titer compared to the group receiving vector DNA+XIAP and DNA-NC alone This group (NC protein + montanide/CpG) also showed a slightly higher anti-body titer compared to the group received DNA-NC + NC protein and XIAP However, the highest SARS-CoV-spe-cific antibody response was detected in mice immunized with a combination of DNA-NC, protein and montanide/ CpG (Fig 3)
Combination of DNA, recombinant protein and XIAP induce higher level of CD8+T-cell immune responses
To assess whether vaccination with nucleocapsid increases cell-mediated immune responses, splenocytes and fresh blood from immunized mice was retrieved, stimulated, and stained for surface CD4 and CD8+T cells as well as intracellular interferon gamma The level of IFN-γ produc-ing CD4+ T cell in fresh blood (Fig 4) and splenocytes (data not shown) from immunized mice did not demon-strate a significant CD4+T cell response against the
SARS-Antibody titers were determined in mice (n = 4) two weeks after the last immunization
Figure 3
Antibody titers were determined in mice (n = 4) two weeks after the last immunization The 96-well plates were coated with SARS-NC protein and mouse sera were serially diluted in wells for the endpoint titration of anti-NC antibody Results are
shown as mean concentration ± S.D The symbol * indicates a significant difference (P = 0.01–000.1) compared with all other groups The symbol † indicates a significant difference (P ≤ 0.001) when compared to animals immunized with DNA+XIAP and DNA-NC alone
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
DNA+XIAP DNA-NC NC p ro tein +
Cp G/M o ntanid e
DNA-NC + NC
p ro tein +
M o ntanid e/Cp G
DNA-NC + NC
p ro t ein + XIAP
∗
†
†
Trang 6NC protein However, the group receiving DNA-NC,
protein and montanide/CpG demonstrated higher levels
of IFN-γ producing CD4+ T cells
Splenocytes were also stimulated with NC protein, and
CD4 lymphocyte proliferation was performed with
tritiated thymidine However, a high T-cell proliferation
was not detected with this assay (data not shown)
Cell-mediated immune responses were evaluated by
intra-cellular cytokine staining The group receiving DNA-NC +
NC protein and Montanide/CpG elicited higher levels of
CD8+T-cells to nucleocapsid in comparison to groups
receiveing DNA-NC or NC protein plus adjuvant
How-ever, the highest NC-specific CD8+T-cell response was
detected in both splenocytes (data not shown) and fresh
blood (Fig 5) in mice that received the DNA construct,
recombinant NC protein and adjuvant XIAP
To confirm the results obtained by intracellular cytokine staining, we performed an IFN-γ ELISPOT assay to meas-ure NC-specific T-cell responses of splenocytes from immunized mice The groups DNA+XIAP, DNA-NC and
NC protein + montanide/CpG did not show a high number of spot forming cells (SFC) Potent IFN-γ responses were observed in mice immunized with combination of DNA-NC+NC protein and adjuvants (Fig 6) However, following substitution of adjuvant montanide/CpG with XIAP, SFCs were more than two
fold higher (p = 0.01) Although, IFN-γ may be produced
by both antigen-stimulated CD4+ and CD8+ T cells, most likely the observed IFN-γ response was generated by effec-tor CD8+ T-cells, since flow cytometry demonstrated CD8+ T cells as the main producers of IFN-γ in this study
SARS-CoV-NC specific CD4+ T cell responses in mice immunized with the candidate vaccines
Figure 4
SARS-CoV-NC specific CD4+ T cell responses in mice immunized with the candidate vaccines Fresh peripheral blood cells were cultured, stimulated with NC protein and stained for CD4, CD3 and IFN-γ Flow cytometry was used to analyse the NC-specific CD4+T cell response A negative control (without stimulation) and a positive control (phorbol myristate acetate + ion-omycin) were included to control for the spontaneous production of IFN-γ(data not shown) Results are shown as mean ± S.D
The symbol † indicates a significant difference (P < 0.05) compared with the control group (DNA+XIAP).
0
0.05
0.1
0.15
0.2
0.25
0.3
+Montanide/CpG
DNA-NC+NC protein + Montanide/CpG
DNA-NC + NC protein + XIAP
†
†
Trang 7The SARS epidemic is currently under control However,
the absence of an effective therapeutic agent against this
lethal virus, compounded by the threat of its
re-emer-gence, has triggered research efforts to develop an effective
vaccine Previous studies indicate that the spike protein is
responsible for the binding of the virus to
angiotensin-converting enzyme 2 (ACE2) [25-27] The spike protein
contains epitopes that might elicit neutralizing antibodies
in the host species thus making it a good target for vaccine
development against SARS [28-31] However, mutation of this protein could affect the virulence by allowing the virus to escape from specific immune response[32,33] Other research groups have made efforts to develop vac-cines based on viral nucleocapsids since these viral pro-teins have conserved regions Milich and McLachlan showed that the viral nucleocapsid contains T-cell dependent and independent epitopes Nude (athymic) mice immunized with HBV-nucleocapsid alone develop high titers of IgM, IgG2a and IgG2b antibodies which are
SARS-CoV-NC specific CD8+ T cell responses in mice immunized with the candidate SARS vaccines
Figure 5
SARS-CoV-NC specific CD8+ T cell responses in mice immunized with the candidate SARS vaccines Fresh peripheral blood cells from immunized mice were stimulated with various antigens and stained for CD8, CD3 and IFN-γ with labeled mono-clonal antibodies After staining, flow cytometry was used to analyze the NC-specific CD8+ T cells A negative control (with-out stimulation) and a positive control (phorbol myristate acetate + ionomycin) were included to control for the spontaneous production of IFN-γ Cells were also stimulated with an irrelevant protein, HIV-1 gp120 (data not shown) A: Dot plots show results from individual representative animals from each group of mice B: Results are shown as mean ± S.D The symbol *
indi-cates a significant difference (P = 0.01–000.1) compared to all other immunized groups The symbol † indiindi-cates a significant dif-ference (P = 0.026) compared to the control group.
A
B: DNA-NC
D: DNA-NC + NC protein + Montanide/CpG E: DNA-NC + NC protein + XIAP
C: NC protein + Montanide/CpG A: DNA alone + XIAP
B
0 0.2 0.4 0.6 0.8 1 1.2 1.4
DNA + XIAP DNA-NC NC protein +
Montanide/CpG
DNA-NC + NC protein + Montanide/CpG
DNA-NC + NC protein + XIAP
∗
†
Trang 8the predominant antibodies in Th1 responses [34] There
is evidence that the specific structure folding of viral
nucleocapsids is responsible for its high immunogenicity
[35]
The success of immunization depends on several factors,
such as type of antigen, route of administration and usage
of adjuvants Mittal et al showed [36] that mice
immu-nized intramuscularly, intraperitoneally or
subcutane-ously have higher antibody titers than mice immunized
orally or intranasally In this study, mice were immunized
subcutaneously as this route of administration has been
used successfully in the past [37-39]
We promoted the immune responses with adjuvants
montanide ISA-51/CpG or XIAP Montanide is a mineral
oil based adjuvant that increases the immune response
non-specifically[40,41] It has been tested in clinical trials
and it has a good reactogenicity profile, making it an ideal
adjuvant for human use [42-44] In an HIV vaccine
candidate study, we showed that montanide can induce strong antibody titers against HIV-1 structural genes (gp120, gag and pol) CpG is also among the most fre-quently used experimental adjuvants; this adjuvant stim-ulates dendritic cells through Toll-like receptor 9 (TLR9), inducing cell maturation and enhancing antigen presenta-tion and Th1 responses [45-47] The combinapresenta-tion of montanide and CpG was investigated in light of a recent study demonstrating that this combination is more effec-tive than the use of any of the adjuvants alone [48] A group of mice received XIAP as adjuvant based on the finding by Kim et al that mice immunized with DNA encoding XIAP exhibit a strong cell mediated immune response against melanoma Kim et al hypothesize that this strong response may be due to increased survival of dendritic cells or T cells in vivo [16,17]
Nucleocapsid has a fundamental role in the viral life-cycle and could be a potential target for enhancing the immune responses It is also of interest as a particulate carrier for
Figure 6
The number of IFN-γ producing cells was measured by an ELISPOT assay The plates were coated with an anti-mouse IFN-γ antibody The cells were cultured in the presence of recombinant NC protein or an irrelevant antigen (gp120 protein) NC-specific IFN-γ were detected as described in Materials and Methods The mean ± S.D is shown for each group The symbol * indicates a significant difference (P = 0.01–0.001) between the indicated group and all other immunized groups The symbol † indicates a significant difference (P < 0.05) between the indicated group and the control group (DNA+XIAP).
0
50
100
150
200
250
300
350
400
450
DNA+XIAP DNA-NC NC protein +Montanide/CpG DNA-NC+NC
protein+Montanide/CpG
DNA-NC+NC protein+XIAP
∗
†
Trang 9conserved CD8+T-cell epitopes that might be suitable for
the development of an effective vaccine for SARS-CoV
In order to characterize specific immune responses in our
candidate SARS vaccines, we used a recombinant protein
expressed in bacteria for in vitro assays to detect CD4+
and CD8+T-cell responses, while the vaccine candidates
contained a recombinant protein expressed in CHO cells
Ideally, peptides are used to stimulate CD8+effector
responses, however, this is not yet feasible since NC CTL
epitopes are not yet characterized in this strain of mice It
is likely that VLPs are processed by antigen presenting cells
and the epitopes presented in an MHC I context, as
suggested by the increased CD8+ T-cell responses
observed post-vaccination
Several studies have assessed the SARS-CoV-NC protein as
a candidate vaccine For instance, Wang et al [49] showed
a low proliferative response to NC in BALB/c mice that
receive a DNA vector expressing NC protein A weak
CD4+T cell response was also observed in our study Two
more studies analyzed humoral and cell-mediated
immune responses in mice immunized with DNA
vac-cines expressing NC [50,51] Kim at al showed that
link-age of NC protein to calreticulin increased humoral and
cellular immune responses in vaccinated mice compared
to mice receiving DNA-NC alone We did not detect a high
level of CD8+ T cell immune response in mice immunized
with DNA-NC or NC protein alone However, the
immunogenicity of our candidate DNA vaccine encoding
NC was improved with the co-administration of the
recombinant nucleocapsid protein and adjuvants
Zhu et al show a high level of antibody titer in mice after
three injections of DNA-NC Surprisingly, we did not
detect a high level of antibody titer in mice immunized
with DNA-NC alone
In summary, our results indicate that immunization with
different adjuvants could influence the type of immune
response Mice that received DNA, protein and
monta-nide/CpG showed a high level of specific antibody titer
against NC However, vaccination with combinations of
DNA-NC, recombinant NC protein and XIAP may add
breadth to cell-mediated immune responses These results
suggest a novel approach to produce an effective vaccine
against SARS infection
Abbreviations
XIAP: X-linked inhibitor of apoptosis
NC: Nucleocapsid
Acknowledgements
We thank the personnel in the animal facility at University of Ottawa for their assistance We are grateful to Drs Katrina Gee and Neera Malik for critically reading the manuscript.
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